Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Contemporary chemotherapeutic regimens confer an overall probability of cure approaching 90%.1 However, ALL has long been recognized to be biologically heterogeneous, with significant differences in treatment outcome between discrete patient subgroups, distinguishable through the integration of clinical (age, presenting white cell count, central nervous system involvement), leukemia-associated (genetic aberrations), and therapy-related (posttreatment minimal residual disease) parameters.2
The paradigm of risk-adapted therapy in childhood ALL is premised on the availability of effective curative treatment approaches for each patient subgroup. Although this approach has successfully ameliorated the prognostic impact of some previously adverse disease features such as t(1;19),3 other patient groups continue to have a markedly inferior outcome even with the most intensive current therapy, including hematopoietic stem cell transplantation. These include patients with low hypodiploid,4 Philadelphia-like (Ph-like),5 or infant mixed lineage leukemia-rearranged ALL (MLLr-ALL).6 Similarly, effective treatment options are lacking for many patients with relapsed childhood ALL, particularly early relapses with marrow involvement.7
BCL-2 family proteins regulate the intrinsic apoptotic pathway by integrating diverse prosurvival or proapoptotic intracellular signals.8 In healthy cells, proapoptotic BAX and BAK are kept in check by their prosurvival relatives (BCL-2, BCL-X, BCL-W, MCL-1, A1). Cellular stress signals, such as DNA damage–induced TP53 activation, trigger proapoptotic BH3-only proteins (such as BIM, PUMA)9 to neutralize the prosurvival BCL-2 proteins or directly activate BAX or BAK, thus initiating apoptosis.
The intrinsic apoptotic pathway mediates the cytotoxic activity of most chemotherapeutic agents. Mutations that compromise the integrity of this pathway, or upstream stress signaling (in particular, the dysregulation of TP53), have been recognized as major contributors to chemoresistance and treatment failure.10,11 In ALL, BCL-2 upregulation is associated with slow early response to therapy,12 although BCL-2 levels do not predict overall outcome.13,14 Clinical resistance to corticosteroids, an independent predictor of adverse outcome in ALL and feature of poor prognosis ALL subtypes including infant MLLr-ALL,15 is associated with reduced proapoptotic BIM induction and high MCL-1 expression.15⇓-17 Conversely, a rapid early response is associated with upregulation of BIM.12 Notably, the cooccurrence of single-nucleotide polymorphisms resulting in BIM inactivation and increased prosurvival MCL-1 also predicts markedly inferior outcomes following contemporary ALL treatment.18
BH3-mimetics are novel therapeutic agents designed to circumvent the apoptotic dysregulation that confers resistance to standard chemotherapy, by directly targeting prosurvival BCL-2 proteins. Navitoclax (ABT-263), which binds BCL-2, BCL-XL, and BCL-W with nanomolar affinity,19 has potent activity in chronic lymphocytic leukemia (CLL), and in phase 1 trials induced objective responses in 30% of heavily pretreated patients.20 Dose escalation was curtailed by on-target thrombocytopenia due to BCL-XL inhibition.20,21 The discovery that BCL-2 inhibition accounts for the cytotoxic activity of navitoclax in CLL spawned development of the BCL-2-selective BH3-mimetic venetoclax (ABT-199).22 Without the thrombocytopenia that limited navitoclax dosing, substantially higher venetoclax exposures have been achievable, resulting in objective response rates of ∼80% in phase 1/2 CLL trials.23,24
Preclinical studies in childhood ALL xenografts with navitoclax, and its preclinical predecessor ABT-737 (which has identical BH3-mimetic properties),25 have demonstrated potent single-agent activity as well as synergistic cytotoxicity in combination with standard chemotherapy.26⇓-28 Although ALL blasts generally express significantly higher levels of BCL-2 compared with normal lymphoid precursors,29,30 they also express significant levels of BCL-XL, in contrast to CLL, where BCL-XL is dominated by BCL-2.31 We have undertaken detailed studies exploiting a BH3-mimetic toolkit, to determine whether selective inhibition of BCL-2 is potentially an effective strategy for treating childhood ALL, given the superior tolerability of venetoclax compared with navitoclax, or conversely, if inhibition of BCL-XL contributes significantly to the observed efficacy of navitoclax in high-risk childhood ALL xenografts.
Materials and methods
Xenografts and in vivo drug treatments
All experimental studies were conducted with approval from the Animal Care and Ethics Committee of the UNSW (Sydney, Australia). Procedures for establishing continuous xenografts from childhood ALL biopsies in immune-deficient NOD/SCID (NOD.CB17-Prkdcscid/SzJ) or NOD/SCID, IL-2 receptor γ−negative (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, NSG) mice are described elsewhere.32 All xenografts used in this study have been previously described and were used at between the third and fifth passage.27,33 Their clinical features are summarized in supplemental Table 1, available on the Blood Web site. Venetoclax (obtained from AbbVie) was administered orally at a dose of 100 mg/kg/d for 21 days.
Leukemia engraftment and progression were assessed in groups of 6-10 female mice, each weighing 20 to 25 g, by weekly enumeration of the human CD45+ cells in proportion to peripheral blood (%huCD45+).32 Individual mouse event-free survival (EFS) was calculated as number of days from treatment initiation until %huCD45+ reached 25%, computed by interpolating between bleeds directly preceding and following events, assuming log-linear growth. Efficacy of drug treatment was evaluated by leukemia growth delay (calculated as T − C, the difference between median EFS of vehicle control [C] and drug-treated [T] cohorts), and an objective response measure (ORM), as described previously (detailed description in supplemental Methods and supplemental Table 2).34
Immunoblotting, RNA extraction, and gene expression analysis
Preparation of xenograft lysates, protein analysis by immunoblotting, and RNA extraction, purification, and gene expression analysis have been previously described elsewhere27,31,35,36 Gene expression datasets can be accessed at www.ncbi.nlm.nih.gov/geo (accession number GSE52991). Benjamini and Hochberg’s false discovery rate37 measurement and Smyth’s unadjusted P value38 were used for evaluation of differential gene expressions. Gene expression heatmaps were generated using GenePattern.
In vitro cytotoxicity assays (AlamarBlue)
Xenografts were thawed and resuspended in Quality Biological Serum Free-60 (Quality Biological) supplemented with Flt3-ligand (20 ng/mL), penicillin (100 U/mL), streptomycin (100 µg/mL), and l-glutamine (2 mmol/L), and equilibrated overnight at 37°C, 5% CO2 before addition of various concentrations of venetoclax (range 10 µM to 1 pM), or vehicle control. AlamarBlue (Thermo Fisher Scientific) was added 48 hours after drug addition, and fluorescence was measured (560-nm excitation and 590-nm emission wavelengths) on a Victor X3 Multilabel Plate Reader (PerkinElmer, Waltham, MA) 6 hours thereafter. All readings were calculated with 0-hour background subtraction and normalized as a percentage of control wells.
Coculture in vitro cytotoxicity assays
Xenograft cells were cocultured with human telomerase reverse transcriptase (hTERT)-immortalized human mesenchymal stromal cells (hTERT-MSC; kindly provided by Dario Campana) as described previously.27 Briefly, 50 000 hTERT-MSCs per well were seeded in RPMI 1640 + fetal calf serum 10% in 96-well plates and left overnight to form adherent monolayers. Media were then changed to QBSF-60 (+ penicillin, streptomycin, glutamine, and Flt3-ligand 20 ng/mL). Cryopreserved xenograft cells were thawed, resuspended in this media, and added to hTERT-MSC–lined wells. After 4 hours, navitoclax, venetoclax, A-1113567,39 or A-115546340 alone or in selected combinations were added to triplicate wells to achieve final concentration ranges as per the figure legends. Following 24-hour drug exposure at 37°C in 5% CO2, viability was determined by propidium iodide exclusion using a FACSCalibur flow cytometer, and lethal concentrations, 50% (LC50s) were calculated by nonlinear regression analysis using Graphpad Prism.
Evaluation of drug synergy
Xenograft cells were treated in triplicate with serial 4-fold dilutions (range 0.25 nM to 1 μM) of venetoclax and A-1155463 alone and in a combination matrix that paired every concentration of each drug with the full range of concentrations of the second drug. The predicted additive effect of combined BH3-mimetics was calculated using the Bliss model of fractional independence41 and subtracted from the actual measured combinatorial effect to generate Bliss scores for each combination of drug concentrations. Bliss scores across each combination concentration matrix were summed for quantitative comparison across samples as previously described.40
EFS curves were compared using both log-rank and Gehan-Wilcoxon tests. The latter gives more weight to early events. Pearson’s correlation test was used for all datasets with normal distribution; otherwise Spearman’s correlation test was used. Significance was inferred from tests with P values <.05, except where α was corrected for multiple comparisons (threshold for significance: α/number of comparisons = 0.05/n).
Venetoclax exhibits in vivo efficacy in only a minority of pediatric ALL xenografts
We have previously reported that navitoclax (100 mg/kg/d for 21 continuous days) exhibits broad in vivo efficacy in pediatric ALL, inducing leukemia progression delay >10 days in 27 (87%), and objective responses in 19 (61%) of 31 xenografts representing a range of high-risk ALL subtypes.26,27 To determine if this efficacy could be recapitulated by selectively targeting BCL-2, we initiated similar studies with venetoclax (100 mg/kg/d for 21 continuous days) against a panel of 19 pediatric ALL xenografts, including a representative subset of 16 from the above panel, of which 9 (56%) had achieved objective responses (3 partial responses [PRs], 4 complete responses [CRs], 2 maintained complete responses[ MCRs]) following navitoclax therapy. Overall, the venetoclax panel comprised 4 MLLr-ALL, 5 B-cell precursor (BCP-ALL), 4 BCP-ALL with known Janus kinase (JAK) mutations (JAK-mutated ALL), 4 T-cell (T-ALL), and 2 early T-cell precursor (ETP)-ALL xenografts.
Venetoclax significantly delayed the progression of 11/19 (58%) xenografts tested (Table 1; Figure 1A-E; supplemental Figure 1), but only in 6/19 (32%) was leukemia progression delay >10 days (range −2.6 [ALL-27; P = 1.0] to 27.7 [ALL-17; P < .001]). When stratified by ALL subtype, median leukemia growth delay T − C calculations were 8.2 days for MLLr-ALL (range 1.2–21.0), 10.9 days for BCP-ALL (range 0.9–27.7), 3.8 days for JAK-mutated ALL (range 1.3–10.7), −1.2 days for T-ALL (range −2.6–4.6), and 1.9 days for ETP-ALL (range 1.8–1.9) (Figure 1F; Table 1). Notably, leukemia progression delay associated with venetoclax treatment appeared comparable to previously reported results with navitoclax27 for subgroups such as MLLr-ALL and BCP-ALL (Figure 1F). In contrast, there is a trend suggestive of superior progression delay in the navitoclax-treated T-ALL group, although this difference was not statistically significant (P = .017; corrected for multiple comparisons, α = 0.05, n = 5).
In vivo single-agent venetoclax responses of pediatric ALL xenografts. Responses of representative xenografts from the (A) MLLr-ALL, (B) BCP-ALL, (C) JAK-mutated ALL, (D) T-ALL, and (E) ETP-ALL subpanels treated with venetoclax (100 mg/kg for 21 days, dotted lines) or vehicle control (solid lines); results from individual mice are represented by gray lines, whereas the black lines summarize the outcome for each cohort. In each case, the left panels represent the %huCD45+ of individual mice over time, whereas the right panels show the proportion of mice remaining event free. (F) Leukemia growth delay (T − C) of ALL xenograft subtypes following treatment with navitoclax or venetoclax. Each data point represents the median cohort leukemia growth delay for each xenograft; the horizontal bar represents the median for each ALL subtype. Data from the navitoclax cohort have been published previously27 and are included here for comparison. Statistical comparison between cohorts treated with navitoclax vs venetoclax was by unpaired Student t tests corrected for multiple comparisons using the Bonferroni method. (G) “COMPARE-like” plot of the difference between the median ORM of xenografts shown in Table 1 and the midpoint response (which corresponds to a score of 5). Bars to the right or left of the midpoint represent objective responses or nonobjective responses, respectively. Xenografts achieving a PR (median ORM 6), CR (median ORM 8), or MCR (median ORM 10) classify as responders, whereas those with progressive disease (PD1, median ORM 0; or PD2, median ORM 2) classify as nonresponders.
When responses were evaluated using clinically based criteria as previously described,34 objective responses were observed in 5/19 (26%) xenografts, with 3 CRs and 2 PRs (Table 1). Figure 1G represents the venetoclax responses of each xenograft in a “COMPARE-like” format.34 In agreement with the observed delays in leukemia progression, no significant differences were observed between ALL subtypes (Kruskal-Wallis, P = .24). A complete summary of results is provided in supplemental Figure 1 and supplemental Table 3. Of note, only 1/177 (0.56%) mice experienced toxicity-related morbidity following venetoclax treatment.
Overall, in vivo responses to venetoclax in a comparable test panel appeared less pronounced, were mostly less sustained, and were observed in a markedly smaller subset of ALL xenografts, compared with our previously published experience with navitoclax.27
In vivo responses of pediatric ALL xenografts to venetoclax
High BCL-2 and low BCL-XL is associated with in vivo venetoclax response
We next sought to better understand the determinants of venetoclax response, to identify potential clinical biomarkers that identify candidates for venetoclax therapy, or conversely, patients who might respond better to navitoclax. Therefore, we compared BCL-2 family mRNA and protein expression in xenografts classified as responders (n = 5) vs nonresponders (n = 14).
Of 18 genes evaluated by microarray analysis, only BCL2L1 (encoding for BCL-XL/XS) approached significance in being upregulated in nonresponders vs responders (Figure 2A; P < .01), although the difference was not significant when corrected for multiple comparisons. There was correspondingly higher differential BCL-XL protein expression (Figure 2B-C; P = .014) in nonresponders, which again was not significant when corrected for multiple comparisons. Conversely, response to venetoclax was positively and significantly associated with higher BCL-2 protein expression (Figure 2B-C; P = .0072), although there was clear overlap in expression levels between groups. There was no significant difference in BAK, BAD, BIM, or MCL-1 mRNA, or protein expression between the 2 groups (Figure 2A-C).
Evaluation of BCL-XL and BCL-2 as potential determinants of in vivo venetoclax responses. Xenografts were stratified into venetoclax in vivo responders and nonresponders. (A) Microarray analysis of the BCL2 family of genes with each row representing a gene. Prosurvival genes are listed at the top (in red labels), with BCL2L1 (which encodes BCL-XL) highlighted with an arrow. There are 3 groups of proapoptotic genes, encoding BCL-2 family proteins bearing all 4 BCL-2 Homology (BH) domains (BAX/BAK-like in blue labels), the BH3 domain only (purple labels), or BH3 and BH2 domains (green labels). The colors in the heatmaps represent the relative expression per gene across all samples. Red indicates relative high expression, and blue indicates relative low expression. (B) Immunoblot analysis of BCL-2 family protein expression in all 19 xenografts. (C) Protein expression of BCL-2 family members in 19 xenografts was quantified using Versadoc and calculated relative to the expression of actin within each sample and normalized to HL-60. Each dot represents an average of 3 biological replicates of each xenograft. Groups were compared using Mann-Whitney tests. With Bonferroni correction for multiple comparisons (n = 6), P < .0083 for statistical significance.
When analyzed against leukemia T − C values and corrected for multiple comparisons, there were positive correlations only with the ratios of BCL-2/BCL-XL protein (Figure 3C; R2 = 0.47; P = .001) and mRNA (Figure 3F; R2 = 0.39; P = .004) expression, and BCL-2 protein levels (Figure 3B; R2 = 0.47; P = .0013). The correlations between leukemia growth delay and BCL-XL mRNA expression (Figure 3D; R2 = 0.34; P = .008) and BCL-2:MCL-1 protein ratios (supplemental Figure 2; R2 = 0.35; P = .008) only approached significance when corrected for multiple comparisons. Almost identical correlations were obtained using T/C, rather than T − C, values of in vivo venetoclax efficacy (supplemental Figure 3).
Correlations between in vivo venetoclax sensitivity and basal BCL-2/BCL-XL expression or in vitro venetoclax responses. (A-F) Basal protein or mRNA expression of BCL-XL, BCL-2, or the ratio of BCL-2:BCL-XL is plotted against leukemia growth delay (T − C). With Bonferroni correction for multiple comparisons (n = 8), P < .00625 for statistical significance. Each data point represents 1 xenograft. (G-H) In vitro LC50 values by AlamarBlue or coculture assays are plotted against leukemia growth delay (T − C). With Bonferroni correction for multiple comparisons (n = 2), P < .025 for statistical significance.
Taken together, these results indicate that higher levels of prosurvival proteins such as BCL-XL and MCL-1 that are not directly targeted by venetoclax undermine its antileukemic efficacy, whereas high BCL-2 expression may identify ALL patients more likely to benefit from venetoclax therapy. However, it is also evident, given the large overlap between groups in their expression of individual BCL-2 family proteins or ratios of selected proteins, that their quantification is insufficiently reliable as clinical biomarkers.
In vitro single-agent venetoclax sensitivity predicts in vivo venetoclax responses in pediatric ALL xenografts
We next investigated the relationship between in vitro venetoclax sensitivity and in vivo responsiveness in the panel of ALL xenografts. All 19 xenografts were treated with venetoclax (concentration range 1 pM to 10 μM) in vitro for 48 hours, and viability was assessed using the AlamarBlue mitochondrial activity assay. When corrected for multiple comparisons, there was a significant inverse correlation between in vitro venetoclax LC50 and leukemia growth delay (Figure 3G; R2 = 0.53; P = .0004). Median venetoclax LC50 was also significantly lower (P = .0007) in responder xenografts (median LC50 1.8 nM; range 0.3-6.8 nM), compared with nonresponders (median IC50 0.75 μM; range 5.0 nM to 4.5 μM) (Figure 4A; supplemental Figure 4). However, again some overlap was observed between the groups.
In vitro response to BH3-mimetics in pediatric ALL xenografts. (A) Summary of the mean venetoclax LC50 at 48 hours of each xenograft determined by AlamarBlue assay. Data are segregated by venetoclax in vivo response. Bar represents the median LC50 of each cohort. Groups compared by Mann-Whitney test. (B) ALL xenografts cocultured with hTERT-MSCs were treated (concentration range 1 nM-4 μM) with navitoclax (n = 24), venetoclax (n = 24), A-1113567 (n = 23), or A-1155463 (n = 24) alone, or an equimolar 50:50 mixture of venetoclax and A-1155463 (n = 24). Viability was evaluated using propidium iodide exclusion by flow cytometry, and LC50s calculated by nonlinear regression in Graphpad Prism are summarized here. A range rather than scale is depicted for LC50 > 10 μM, as these correspond to extrapolated rather than measured values. Wilcoxon matched-pairs signed rank test was used for statistical comparison between groups. P values are represented as follows: *P ≤ .05; **P ≤ .01; ***P ≤ .001; ****P ≤ .0001. (C) Sensitivity to navitoclax correlates significantly with sensitivity to venetoclax alone or a 50:50 mixture of venetoclax and A-1155463, but not A-1155463 alone. A-1155463 LC50 could not be calculated for the 2 samples with the highest navitoclax LC50. Pearson correlation coefficients are listed. (D) Sensitivity to navitoclax, venetoclax, A-1113567, or A-1155463 alone, or the venetoclax and A-1155463 50:50 mixture, by ALL subtype. (E) In MLLr-ALL, sensitivity to navitoclax correlates strongly with venetoclax sensitivity.
ALL blasts survive only briefly in vitro, implicating a significant role for the microenvironment in sustaining their survival. Notably, such factors may impact sensitivity to BH3-mimetics.42 We have previously demonstrated that navitoclax sensitivity of pediatric ALL xenografts in an in vitro coculture assay predicts in vivo responses.27 We thus evaluated the venetoclax sensitivity of our xenograft panel cultured on a hTERT-MSC feeder layer. As the high background metabolic activity of hTERT-MSCs precludes the Alamar Blue assay, viability of cocultured xenograft cells was assessed by flow cytometry. Notably, there was good correlation between in vitro sensitivity determined by each method (supplemental Figure 5) and LC50 derived from the coculture assay also correlated with leukemia growth delay (Figure 3H).
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